The long-term objectives of this application are 1) to understand how mitochondrial genes are expressed in human cells and how this expression is regulated; 2) to gain an insight into the control of assembly of the oxidative-phosphorylation apparatus of the cell in relationship to functional, developmental and tissue- specific parameters; 3) to learn how to manipulate at will the mitochondrial gene complement of a cell. Specifically, the present proposal aims at investigating vivo or vitro using isolated organelles or submitochondrial systems, the mechanisms whereby the differential rate of synthesis of mitochondrial rRNA and mRNA is achieved, the structure and function of one of the mitochondrial RNA processing enzymes, i.e., the RNAse P, with particular reference to its RNA component, and the molecular basis of translational control in human mitochondria. In addition, it is planned to clone and to investigate the structure and expression of nuclear genes specifying key enzymes of the oxidative-phosphorylation apparatus, i.e., the ADP/ATP translocase and the NADH dehydrogenase. Finally, it is planned to further develop the work already started in this laboratory for mitochondria-mediated and mitochondrial DNA- mediated transformation of human cells. The achievement of the above aims will have significant implications for understanding how mitochondria work in different human tissues and what is the molecular basis of mitochondrial defects in the ever-growing class of "mitochondrial" diseases. Furthermore, the possibility of introducing new mitochondrial genes into a cell opens exciting prospects for diagnosis and cure of genetic defects of mitochondrial DNA.